Cystic fibrosis is a lethal genetic disease afflicting approximately 30,000 individuals in the United States. Since 1 in 2500 Caucasians is born with cystic fibrosis, it is the most common lethal, recessively inherited disease in that population. This inherited disorder impairs epithelial ion transport, particularly that of chloride. Cystic fibrosis affects the secretory epithelia of a variety of tissues, altering the transport of water, salt and other solutes into and out of the blood stream. In particular, the ability of epithelial cells in the airways, pancreas and other tissues to transport chloride ions, and accompanying sodium and water, is severely reduced in cystic fibrosis patients, resulting in respiratory, pancreatic and intestinal ailments. The principle clinical manifestation of cystic fibrosis is the resulting respiratory disease, characterized by airway obstruction due to the presence of thick mucus that is difficult to clear from airway surfaces. This thickened airway liquid contributes to recurrent bacterial infections and progressively impaired respiration. Death may occur in severe cases because of chronic lung infections, especially by Pseudomonas aeruginosa, which cause a slow decline in pulmonary function.
One current treatment for CF patients focus on controlling the symptoms of infections through antibiotic therapy and promoting mucus clearance by use of postural drainage and chest percussion. However, even with such treatments, frequent hospitalization is often required as the disease progresses. Thus, long-term therapies are needed for these patients.
Exposure to beryllium in the form of the metal, metal oxide, or metal alloy continues to be a major public health issue, and approximately 800,000 people are currently at risk for development of chronic beryllium disease (CBD). The pulmonary lesion in CBD is histologically indistinguishable from that observed in sarcoidosis, but the etiology of sarcoidosis remains unknown (Newman et al. 1998). The presence of antigen presenting cells (APC) expressing class II molecules is required for CD4+ T cells from CBD patients to proliferate in the presence of beryllium in vitro (Saltini et al. 1989). Recent studies also suggest that susceptibility to developing CBD is associated with particular alleles of the class II major histocompatibility complex (MHC) molecule, HLA DPB1 Glu69 (Richeldi et al. 1993). In both CBD and sarcoidosis, the development of granulomatous inflammation is dependent upon activation of T cells (Saltini et al. 1990; Thomas & Hunninghake 1987). Once T cell activation occurs in the lung of CBD patients, Th1-type cytokines, such as interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor (TNF), are produced (Tinkle et al. 1997). The release of inflammatory cytokines and the Th-1 response results in macrophage accumulation, activation, and the development of granulomatous inflammation.
Be-exposed subjects have normal lung histology and lack evidence of an immune response to Be exposure in vitro. Over time, approximately 5-16% of Be-exposed subjects progress to beryllium-sensitization (BeS). BeS is characterized by normal lung histology and lung function, but with the presence of Be-specific T cells in the blood. When these cells are placed in culture, BeS peripheral blood mononuclear cells exposed to Be salts will proliferate as shown by their ability to incorporate the DNA-specific precursor, tritium labeled (3H)-thymidine (3HTdR). Thymidine uptake is measured using the beryllium lymphocyte proliferation test (BeLPT) as described by Mroz et al. [ref Mroz et al. J Allergy Clin Immunol 88:54, 1991].
Approximately 16% of BeS subjects progress to CBD characterized by the presence of Be-specific T cells in both the blood and bronchoalveolar lavage cells and by the presence of non-caseating granulomatous lung inflammation. These Be-specific blood and lung T cells are also demonstrated by their ability to incorporate 3HTdR in the BeLPT. In some instances, granulomatous inflammation in CBD can progress to increasing fibrosis with worsening lung function and death.
Currently, there is no known cure for CBD. The symptoms of CBD, principally the symptoms associated with worsening lung function, are treated by anti-inflammatory steroids and in some instances where steroid use fails, by the anti-inflammatory drug, methotrexate [ref Maier et al Beryllium Disease. In Rom W N ed. Environmental and Occupational Medicine. Philadelphia, Lippincott-Raven, 1998; pp 1017-1031].
There are approximately 50 known ATP-binding cassette (ABC) transporters in humans, and there are currently about 13 genetic diseases associated with defects in 14 of these transporters. The most common genetic disease conditions include cystic fibrosis, Stargardt disease, age-related macular degeneration, adrenoleukodystrophy, Tangier disease, Dubin-Johnson syndrome and progressive familial intrahepatic cholestasis. At least 8 members of this family are involved in the transport of a variety of amphipathic compounds, including anticancer drugs and some appear to contribute to the resistance of cancer cells to chemotherapy. (Gottesman M M, Ambudkar S V, “Overview: ABC transporters and human disease.” J Bioenerg Biomembr 2001, 33(6):453-8.). ABC transporters are found in all known organisms, and approximately 1,100 different transporters belonging to this family have been described in the literature. The family is defined by homology within the ATP-binding cassette (ABC) region. Most family members also contain transmembrane domains involved in recognition of substrates, which are transported across, into, and out of cell membranes, but some members utilize ABCs as engines to regulate ion channels.
Two different integral glycoproteins, the 170 kD P-glycoprotein (P-gp) and the 190 kD multi-drug resistance protein (MRP), are involved in the acquisition of multi-drug resistance phenotypes in cancer cells. Even though they are members of the ABC superfamily, the primary structures are quite different, only about 15% of the amino acids are identitical. Nevertheless, MRP and P-gp confer resistance to a similar profile of chemotherapeutic agents and play a similar role in the acquirement of multi-drug resistance. Recently, MRP demonstrated the ability to transport the cysteinyl leukotriene, leukotriene C4 (LTC4) (Ding G Y, Shen T, Center M S. Multidrug resistance-associated protein (MRP) mediated transport of daunomycin and LTC4 in isolated plasma membrane vesicles. Anticancer Res 1999; 19:3243-8.), and other glutathione conjugates, suggesting that MRP has a function different from P-gp. MRP is an ATP-dependent glutathione S-conjugate carrier (GS-X pump) and is present in membranes of many, if not all, cells. Overexpression of MRP in tumor cells contributes to resistance to natural product drugs and oxyanions
In cystic fibrosis, defective chloride transport is generally due to a mutation in a chloride channel known as the cystic fibrosis transmembrane conductance regulator (CFTR; see Riordan et al., Science 245:1066-73, 1989), another member of the ABC transporter family. CFTR is a linear chloride channel found in the plasma membrane of certain epithelial cells, where it regulates the flow of chloride ions in response to phosphorylation by a cyclic AMP-dependent kinase. Many mutations of CFTR have been reported, the most common of which is a deletion of phenylalanine at position 508 (.DELTA.F508-CFTR), which is present in approximately 70% of patients with cystic fibrosis. A glycine to aspartate substitution at position 551 (G55 ID-CFTR) occurs in approximately 1% of cystic fibrosis patients.
In a healthy lung, glutathione (GSH) is present in high concentrations in the epithelial lining fluid (ELF) of the lower respiratory tract, with normal levels in human ELF being more than 200-fold greater than that in plasma. ELF GSH is a major component of the screening process that protects the pulmonary epithelium from oxidants released by inflammatory cells as well as inhaled oxidants. In addition, ELF GSH helps maintain the normal function of the immune components of the pulmonary epithelial host defense system. However, in certain conditions, such as idiopathic pulmonary fibrosis and AIDS patients, a substantial ELF GSH deficiency exists. Oral administration of GSH does not achieve significant elevation of GSH level in the lungs and intravenous administration of GSH is associated with a very short plasma half-life of the molecule. Thus, a problem exists in supplementing GSH by conventional means.
Glutathione (GSH) is a multipurpose mono-thiol compound. Pure GSH forms a flaky powder that retains a static electrical charge, due to triboelectric effects, that makes processing difficult. Glutathione is a strong reducing agent, so that autooxidation occurs in the presence of oxygen or other oxidizing agents.
In synthesizing GSH in the body, cysteine, a thiol amino acid is required. Since oral administration of glutathione is ineffective, prodrugs or precursor therapy have been advocated. Administration of cysteine, or a more bioavailable precursor of cysteine, N-acetyl cysteine (NAC) was suggested. While cysteine and NAC are both, themselves, oxygen scavengers, their presence competes with GSH for resources in certain reducing (GSH recycling) pathways. Since GSH is a specific substrate for many reducing pathways, the loading of a host with cysteine or NAC may result in less efficient utilization or recycling of GSH. Thus, cysteine and NAC are not ideal GSH prodrugs to solve a deficiency in GSH. Thus, while GSH may be degraded, transported as amino acids, and resynthesized in the cell, there may also be circumstances where GSH is transported into cells without degradation; and in fact the administration of cysteine or cysteine precursors may interfere with this process. Thus, loading up on the precurser products is also a problem.
A number of disease states have been specifically associated with reductions in GSH levels. Depressed GSH levels, either locally in particular organs, or systemically, have been associated with a number of clinically defined diseases and disease states. These include HIV/AIDS, diabetes and macular degeneration, all of which progress because of excessive free radical reactions and insufficient GSH. Other chronic conditions may also be associated with GSH deficiency, including heart failure and coronary artery restenosis post angioplasty.
Diabetes afflicts 8% of the United States population and consumes nearly 15% of all United States healthcare costs. HIV/AIDS has infected nearly 1 million Americans. Current therapies cost in excess of $20,000 per year per patient, and are rejected by, or fail in 25% to 40% of all patients. Macular degeneration presently is considered incurable, and will afflict 15 million Americans by 2002.
Currently, there is no known cure for CBD. The symptoms of CBD, principally the symptoms associated with worsening lung function, are treated by anti-inflammatory steroids and in some instances where steroid use fails, by the anti-inflammatory drug, methotrexate [ref Maier et al Beryllium Disease. In Rom W N ed. Environmental and Occupational Medicine. Philadelphia, Lippincott-Raven, 1998; pp 1017-1031].
Studies have demonstrated insufficient GSH levels are linked to these diseases. Newly published data implies that diabetic complications are the result of hyperglycemic episodes that promote glycation of cellular enzymes and thereby inactivate GSH synthetic pathways. The result is GSH deficiency in diabetics, which may explain the prevalence of cataracts, hypertension, occlusive atherosclerosis, and susceptibility to infections in these patients.
GSH also functions as a detoxicant by forming GSH S-conjugates with carcinogenic electrophiles, preventing reaction with DNA, and chelation complexes with heavy metals such as nickel, lead, cadmium, mercury, vanadium, and manganese. GSH plays a role in protein folding and deficiencies affect many proteins including surfactins and defensens.